evolution of the spectrin repeat

Jaime Pascual, Jose Castresana and Matti Saraste

Summary

We now know that the evolution of multidomain proteins has frequently involved genetic duplication events. These, however, are sometimes difficult to trace because of low sequence similarity between duplicated segments. Spectrin, the major component of the membrane skeleton that provides elasticity to the cell, contains tandemly repeated sequences of 106 amino acid residues. The same repeats are also present in a-actinin, dystrophin and utrophin. Sequence alignments and phylogenetic trees of these domains allow us to interpret the evolutionary relationship between these proteins, concluding that spectrin evolved from a-actinin by an elongation process that included two duplications of a block of seven repeats. This analysis shows how a modular protein unit can be Accepted

23 May 1997 used in the evolution of large cytoskeletal structures.

Introduction The evolution of eukaryotic cells was associated with the emergence of a large number of new proteins. Nucleotide sequencing has revealed that many of the new eukaryotic proteins are made of structurally and functionally independent domains that are often found in multiple copies on the same protein or in different proteins displaying unrelated functions(’). Low sequence similarities between the homologous domains have made it laborious to analyze the history of the duplication events that occurred during the evolution of multidomain proteins. One example of this group of proteins is spectrin (also called fodrin), the major component of the membrane skeleton.

The membrane skeleton is a semiregular cross-linked network of proteins that lines the cytoplasmic side of the cell membrane, providing elasticity(2). Electron microscopy studies on red blood cells, ghosts and skeletons have revealed a two-dimensional triangulated lattice composed of spectrin tetramers that are cross-linked by junctions containing rods of polymeric actid3). Detergent-extracted skeletons exhibit shape memory, giving support to the notion that the elasticity of red blood cells must be attributed to the skeleton. Since spectrin is the principal constituent of the skeleton it has been suggested that the conformational entropy of spectrin is the basis of the elasticity of the erythrocyte membrane(4,5), although other mechanisms based on enthalpic events have also been proposed to explain the elastic deformability of the skeleton(6). The association of multiple spectrin molecules with F-actin into a two-dimensional meshwork involves a group of accessory proteins such as ankyrin, protein 4.1, adducin and dernatid’). Spec- trin can be seen as the basic component of a system of

structural proteins associated with the plasma membrane, a system that is analogous to the major cytoskeletal structures involving actin, tubulin and intermediate filaments(8).

Spectrin is present in most vertebrate tissues and also in non-vertebrates, including D r o s ~ p h i / a ( ~ , l ~ ) , Acan- fharnoeba(’l), Dycfyosfe/ium(l2) and echinoderms(l3) and, possibly, in higher plants(14). Complete sequences have been determined for cDNAs encoding the a-chain of spectrin from human erythrocytes(15), human fibroblasts(16), chicken brain(17) and Drosophi/a(lo). Information on the primary structure of P-spectrins includes the complete sequences of human erythrocyte(18), human brain(lg), mouse erythrocyte(’O), mouse brain(21) and Dros~ph i /a (~) . New sequence analysis tools and knowledge of the structure of the spectrin repeat inspired us to study in detail the evolutionary history of spectrin and its relation with a-actinin.

Domain composition of spectrin The functional unit of spectrin is a tetramer with an elongated shape of approximate length of 200 nm and width of 5 nm. Two subunits (p and a) are assembled into heterodimers in a head-to-tail mode (the N terminus of the 0- chain interacting with the C terminus of the tr-chain). Two dimers further assemble in a head-to-head manner (the C terminus of the P-chain interacting with the N terminus of the cx-chain), generating tetramers (Fig. 1). Higher oligomers have also been described(22).

Spectrin is a multidomain protein, present in two major isoforms (Fig. 2)(23). In the mammalian erythrocyte isoform, the I-1 subunit contains two calponin-homology (CH) d o m a i n ~ ( ~ ~ , ~ 5 ) at the N-terminal actin-binding region, 16

complete spectrin repeats (b l -b16), a partial spectrin repeat implicated in tetramerization (bl7), and a serine-rich region at the C terminus. The corresponding a-chain has a partial spectrin repeat at the N terminus (ao), 20 complete spectrin repeats (al-a20), a Src-homology 3 (SH3) domain(26] inserted inside the gth repeat (as), and a calmodulin-like domain(27) at the C terminus. The non-erythrocyte (general) isoform, which is abundant in tissues such as brain, contains an additional prolinekerine-rich region and a pleckstrin-homology (PH) domain(28) prior to the serine-rich C-terminal region of the p-chain, and an additional calmodulin-binding region inside the lo th repeat of the a-chain (al0) (Fig. 2).

The serine-rich region at the C terminus of the p-chain is most likely a target for phosphorylation regulating the mechanical stability of erythrocyte Prob- ably it is an unstructured area and it does not show signifi- cant similarity to other protein sequences present in the databases. The same lack of predicted defined structure and homology to other protein sequences is applicable to the proline/serine-rich region. Structural studies using NMR and X-ray techniques have predicted that the PH domain is involved in the binding of spectrin to the membrane via an interaction with phosphatidylinositol bispho~phates(~~). The non-erythroid a-chain has two insertions in comparison to the red cell protein. A 20-residue insertion is present after the C terminus of the SH3 domain within the gth repeat (as) (not shown in Fig. 2). This insertion is predicted to lack any defined structure. A 40-residue insertion is found in the lo th repeat (al0) (Fig. 2). It is involved in the binding to calmodulin and is predicted to fold into an tr-helical conforma- tiod3'). Conversely to what is observed in the other sequences, the calmodulin-binding site in Drosophila a- spectrin (residues 1473-1494, see ref. 32) coincides with the 1 3th repeat (a1 3).

The spectrin repeat The spectrin repeat was originally described as a 106- residue repetitive segment present in tryptic peptide sequences(33). Complete sequences that have been published show that the length of the repeats varies from 99 to

Fig. 1. The spectrin tetramer Spectrin is made up of two subunits, I-1 (red) and ( x (blue] These subunits assemble into heterodimers in an antiparallel head-to-tail fashion (the N terminus of the 1% meets the C terminus of the a ) Two heterodirners make a tetramer through a head-to-head interaction (the C terminus of the I-1 contacts with the N terminus of the (YI (centre)

11 0 residues. The degree of sequence identity between the repeats is low, though it is likely that all of them have similar three-dimensional structures.

Secondary structure prediction based on sequence infor- m a t i ~ n ( ~ ~ ) has suggested that the repeat is made up of three tr-helices (A, B and C) separated by two loop regions (AB and BC). A complete alignment of spectrin repeats helps to discern key conserved features of this domain. The secondary structure is shown in the bottom line of the sequence alignment in Fig. 3. Helix A has a length of 29 residues and contains a highly conserved tryptophan at position A1 7, followed by a hydrophobic residue. This helix also shows a predominance of conserved negatively charged residues at positions A10, A13, A20 and A29. Helix B contains 32 residues with a conserved positively charged residue at position 87 and a conserved negatively charged residue at position B14. A proline residue (colored yellow in Fig. 3) is frequently present inside helix B. Helix C has 29 residues and contains a highly conserved aromatic residue at position C15 that is followed by a hydrophilic residue. Two conserved positively charged residues are present at positions C7 and C25, and the last residue of the repeat is normally a leucine at position C29. The loop regions are characterized by abundant prolines and glycines (yellow and orange respectively, in Fig. 3), which are both typical helix breakers.

Tertiary structure models have been proposed for the folding of the repeat into a left-handed triple-helical bundle(35) or a c~ i led-co i l (~~) . The alignment of the repeats (Fig. 3) shows the heptad pattern in the helical regions, with conserved hydrophobic residues (green positions) in the a and d positions that could be involved in the hydrophobic core of the molecule. This pattern is lost between helices and between repeats, indicative of structurally independent blocks. How- ever, sequence-based predictive methods for ~oiled-coils(3~) do not give a high probability for the folding of the repeat into this type of bundle.

Empirical evidence for independent folding of individual repeats and determination of the boundaries for the repeated domains is based on the expression of cDNAs encoding segments of different length. The correct phase has been determined using protease resistance, circular dichroisrn and fluo- rescence spectroscopy as guide line^(^^-^^). Different phases correspond to the segments containing permuta- tions of successive three-helices (ABC, BCA or CAB). The structure of a homodimer of the 13th repeat (14th if the SH3 domain is considered as a repeat) of Drosophila a-spectrin was solved by X-ray crystallography, leading to a model for the monomeric repeat(35). As mentioned before, the helix C of this particular repeat has been implicated in Drosophila a- spectrin binding to ~a lmodu l in (~~) , although from the sequence point of view it shows no special peculiarity. The solution structure of the monomeric 16th repeat of chicken brain a-spectrin shows that the spectrin repeat folds into a left-handed antiparallel triple helical coiled-coil (J. Pascual et a/., manuscript submitted).

Fig. 2. Domain composition of spectrin isoforms The symbols for the different domains are given at the bottom All spectrin repeats are made up of three helices (A B and C) except b17 which is a partial spectrin repeat consisting of two helices (A and B) and a0 consisting of just one helix (C) The SH3 domain and the calmodulin binding region are inserted between the helix Band C of repeats 9 and 10 respectively

The C-terminal repeat of the P-spectrin (b17, Fig. 2) is constituted by helices A and B. The missing helix C is the lonely helix present at the N terminus of the a-spectrin (a0, Fig. 2). As can be seen in the alignment (line b17/a0 in Fig. 3), the recombined repeat contains the conserved features of a normal intact repeat. The spectrin tetramer is formed through non-covalent interactions between helices A and B

of the partial repeat in the P-chain and the helix C of the partial repeat in the a - ~ h a i n ( ~ l - ~ ~ ) . Remarkably, the crystal structure of the dimeric repeat(35) resembles the proposed structure of the tetramerization site, since helices A and B of one molecule interact with the helix C of the other molecule in the crystal dimer. This tetramerization mechanism is sup- ported by several mutations on those areas which lead to

Fig. 3. Alignment of spectrin repeats. The sequences depicted are from human erythrocyte p (bl-b17) and n (aO-aZ0) spectrin. bl7ia0 indicates the tetramerization repeat. The coloring is by conserved property in >55% of any column. Green, hydrophobic, blue, positive; brown, negative: pink, hydrophilic. All gly (orange) and pro (yellow) positions are colored. In the consensus line, h indicates hydrophobic, + positively charged and - negatively charged conserved residues. The heptad line shows the a and d positions of the helical heptad periodicity. The secondary structure line shows the three helices (A, B, C) (numbered according to ref. 35). The multiple sequence alignment was made using the CLUSTAL W program(57). The figure was prepared with the GDE alignment editor (S. Smith, Harvard) and COLORMASK (J. Thompson, EMBL).

defective oligomerization, causing diseases like hereditary e l l i p to~y tos i s (~~~~5) .

The linker among repeats normally contains only two amino acids. However, there are exceptions. The region between repeat 1 (b l ) and repeat 2 (b2), both in erythrocyte and non-erythrocyte P-chains, comprises 12 residues. The

same applies to the regions between a18 and a19 as well as a19 and a20 of both a-chain isoforms. The connection among repeats separated by a two-residue linker is predicted to be made of a long continuous a-helix composed of helix C of repeat i, helix A of repeat i+l and the two-residue linker region. Intron-exon boundaries within the human

Fig. 4. Phylogenetic trees and evolution scheme (A) Tree containing all repeats from human o-actinin (ac l - ac4) and the distal repeats of human erythrocyte spectrin (b l b2, a19 a20) (Bj Tree of the repeats implicated in the duplication events from human erythrocyte spectrin (b6- b12 a2-a8 a10 a16) Note the seven groups of three repeats always containing one repeat from the 13 chain (red branch) and two repeats from the (I (green branches) (C) Evolution scheme of the spectrin repeat Light green squares denote cr-actinin-like repeats (b l b2, a19 and a2O) dark green squares represent those repeats whose evolution has been traced back through the duplication events (b6-bl2 a2-a8 and alO-al6) red squares indicate the rest of the repeats (b3-b5 b13 b16, a1 a9 a17 and a18j purple rectangles depict the tetrameriration repeat (bl7ia0) The identification of the relationships among o-actinin like repeats (light green squares) and the two duplications of the block of seven repeats (dark green squares) was based on phylogenetic trees of the aligned repeats from human spectrins and cr-actinin made by the neighbor- joining method excluding gaps and using Kimura distance correction for multiple substitutions as implemented in the program CLUSTAL W'571

A ac4

acl

b2

B b9

b10 b8

b12

a1 2 / afi \

b10

b l l

C ALPHA ACTININ

CHS repeats CaM-like

ELONGATED ALPHA ACTININ

ALPHAJBETA ANCESTOR

elongation 1 1111113.

ancestral block

1 duplication

m m m m m m m m m m m m ~ m m

ANCESTRAL

BETA SPECTRIN 1111111-

elongation

ANCESTRAL ,1111118

/ ALPHA SPECTRIN

duplication / !

, m m m m m m m m m m m m m m

elongation

BETA SPECTRIN ALPHA SPECTRIN

genes encoding erythroid spectrin do not correspond to the borderlines of the repe,ated d ~ m a i n ( ~ ~ $ ~ ~ ) , which is in agree- ment with an ancient origin of spectrin.

The spectrin repeat is the major constituent of several proteins belonging to the spectrin family of actin-binding proteins(48) such as ~ p e c t r i n ( ~ ~ ) , a - a ~ t i n i n ( ~ ~ ) , dystrophin and utrophid50). All these proteins contain homologous actin-binding sites made up of two adjacent CH domains. Recently, the most distal repeats of P-spectrin (b l and b2) have been suggested to be involved in actin binding, not as major players but as a stabilizers of the interaction(51). More- over, although only proved for d y ~ t r o p h i n ( ~ * - ~ ~ ) , other repeats could also play a role in that interaction, giving rise to the possibility of multiple low affinity binding sites for actin distributed throughout the different repeats.

Erythrocyte spectrin binds to the integral membrane protein band 3 through ankyrin. The site of interaction of spectrin with ankyrin has been mapped to the amino acid residues 1823-1 865 of human erythroid P-~pec t r in (~~) . This sequence coincides with the helix B of repeat 1 5th of P-spectrin and has the common features of all the B helices. More- over, secondary structure prediction methods(34) propose an a-helical conformation for this sequence. Although this repeat lacks the conserved positive charge at position B7, this is not unique to this repeat. Therefore, the structural basis for ankyrin binding remains unclear.

Evolution of the spectrin repeat The availability of complete sequences for the different members of the spectrin family from several species allows us to trace the evolution of the repeat and the common history of spectrin and a-actinin. Previous reports(56) have made use of dot plots for comparison of these sequences. Here, we use multiple sequence alignments and phylogenetic trees(57) which, together with a precise definition of the boundaries of the different domains present in the members of the family, have allowed us to determine accurately the repeats that underwent successive duplications. Since each repeat sequence is more similar to the corresponding repeat in other species than to any repeat within the same sequence, the most recent event in the evolution of the gene family has been the divergence of species. As a result, we can perform the analysis by exclusively using the human gene sequences for the different members of the spectrin family.

The repeat units in dystrophin and utrophin are the most divergent and difficult to align with the repeats of the other two proteins. Therefore, it is likely that those repeats diverged earlier from the rest, although we cannot exactly indicate the step at which this happened. In the phylogenetic trees that include all repeats from p- and a-spectrin and a- actinin, all a-actinin repeats (acl-ac4) as well as the repeats 1 and 2 of P-spectrin ( b l , b2) and 19 and 20 of a-spectrin (a1 9, a20), form a separate group. Consequently, we have

aligned and made a phylogenetic tree of these repeats and apart, have analyzed the sequences of the other spectrin repeats.

Both a-actinin and spectrin form dimers in the cell. The regions that are in tighter contact include both CH domains, the calmodulin-like domain, the four repeats in a-actinin (acl-ac4) and the most distal repeats of spectrin ( b l , b2, a19, a20). The phylogenetic tree shows the existence of a common ancestor for repeat 1 of a-actinin (acl) and repeat 1 of P-spectrin (b l ) (Fig. 4A). The same is pertinent for the pairs ac2lb2, ac3Ja19 and ac4la20. This evolutionary relationship between repeats may correlate with their function in dimerization. Moreover, the linkers between these repeats do not contain the usual two residues, but rather have ten residues in the case of a-actinin and twelve in the case of spectrin. The observed similarity between these repeats, and the simpler domain organization of a-actinin as compared to spectrin, indicates that spectrin evolved from an ancestral a-actinin.

The elastic properties of spectrin are probably based on the conformational flexibility of the region containing the repeats. These all arose via duplication events from an ancestral repeat, which might have been any of the repeats related to a-actinin. The phylogenetic tree for these 33 repeats (b3-bl7IaO-al8) reveals two major duplication events of a block containing at least seven repeats, since it is possible to detect seven groups with three repeats each. A smaller tree containing these 21 repeats (b6-b12, a2-a8 and a10-a16) (Fig. 4B) shows the seven groups, each one with three repeats, containing one repeat from the 13-chain and two repeats from the a-chain. Although the bootstrap values for these groups are low (due to the short sequence length of the domain), the grouping of correlated repeats in space, i.e. b6Ia2la10, b7/a3/al11 b8la41a12, etc. in the seven instances, firmly supports both duplications of seven contiguous repeats. The existence of ancestral repeats that evolved into P and a repeats indicates the presence of a common ancestor for both chains. On the other hand, assuming the existence of an approximately steady molecular clock, there is a closer relationship between the two o! repeats within each group compared to the [3 repeat, as better observed in the alignment of the seven concatenated repeats and the corresponding phylogenetic trees (data not shown), indicating that the last duplication involved the two blocks of the a-chain. Therefore, it can be concluded that the current 21 repeats have evolved from an ancestral block that underwent two successive duplications whose order and exact boundaries are reflected in the phylogenetic trees.

Taken together, the data support a scenario (Fig. 4C) in which an ancestral a-actinin is the source of the repeats in the spectrin family. In the case of spectrin, the major events can be traced for the terminal repeats (b l , b2, a19, a20) and for 21 of the 33 remaining repeats. The beginning of the process can be described as the elongation of a-actinin by

insertion of a block of seven repeats between ac2 and ac3. The next step is the duplication of that block within the elongated a-actinin and an insertion of the ancestor of the tetramerization repeat (b17/a0) between the blocks. Most likely, the insertion of a transcriptional promoter inside the genomic region coding for the ancestor of the tetramerization repeat caused the splitting of that repeat into the partial repeats b17 and aO, and the separation of spectrin into the ancestral P and u subunits. The ancestral P-chain could have evolved by an elongation process that created the rest of the current repeats. Alternatively, the ancestral a-chain might have elongated via the second duplication of the seven-repeat block and insertion of single repeats, although the phylogenetic trees are also compatible with the possibility that the second duplication of the block occurred before the split into f~ and a subunits, leading to a rather long p/u ancestor.

Conclusion Although the non-repetitive segments of spectrin are implicated in specialized roles, the repeats may have also acquired specific functions through sequence drift. Improved algorithms for sequence relation and pattern identification should shed light on those possible unique roles of single repeats and define the precise evolution of the 12 repeats still unrelated. Moreover, sequences of proteins related to spectrin and a-actinin from protists, fungi or plants might reveal other events in the evolutionary history of these proteins. In particular, it would be possible to depict a more detailed evolutionary scenario if proteins related to the inter- mediates postulated in Fig. 4C are discovered in extant organisms. In the light of numerous ongoing genomic pro- jects, such discoveries may be made in the near future.

Acknowledgements We are grateful to Marko Hyvonen for critically reading the manuscript and very helpful suggestions. For more information about spectrin, check our web pages: (http://www.embl- heidelberg.de/External Info/Saraste/spectrin. html).

References 1 Doolittle, R. (1995) The multiplicity of domains in proteins. Annu. Rev Biochem. 64,287-314 2 Elgsaeter, A,, Stokke, B.T., Mikkelsen, A. and Branton, D. (1986). The molecular basis of erythrocyte shape. Science 234, 121 7-1 222. 3 Shotton, D.M., Burke, B.E. and Branton, D. (1979). The molecular structure of human erythrocyte spectrin. Biophysical and electron microscopic studies. J. Mol. Biol. 131,303-29 4 Svoboda, K., Schmidt, C.F., Branton, D. and Block, S.M. (1992). Conformation and elasticity of the isolated red blod cell membrane skeleton. Biophys. J 63,784-793. 5 McGough, A.M. and Josephs, R. (1990) On the structure of erythrocyte spectrin in partially expanded membrane skeletons. Proc. Natl Acad. Sci. USA 87. 5208-1 2. 6 Vertessy, B. and Steck, T. (1989). Elasticity of the human red cell membrane skeleton. Biophys. J. 55, 255-262. 7 Hitt, A. and Luna, E. (1994) Membrane interactions with the actin cytoskeleton. Curr. Opin. CellBiol.6, 120-130. 8 Bennett, V. and Gilligan, D.M. (1993). The spectrin-based membrane skeleton

and micron-scale organization of the plasma membrane. Annu. Rev Cell Biol. 9. 27-66. 9 Eyers, T.J., Brandin, E., Lue, R.A., Winograd, E. and Branton, D. (1992). The complete sequence of Drosophila beta-spectrin reveals supra-motifs comprising eight 106-residue segments. Proc. NatlAcad. So. USA 89, 6187-91 10 Dubreuil, R.R. eta/. (1989). The complete sequence of Drosophila alpha- spectrin: conservation of structural domains between alpha-spectrins and alpha- actinin. J. CellBiol 109, 2197-205. 11 Pollard, T. (1984). Purification of a high molecular weight actin filament gelation protein from Acanthamoeba that shares antigenic determinants with vertebrate spectrins. J. Cell Biol. 99, 1970-1 980. 12 Bennett, H. and Condeelis, J. (1988). Isolation of an immunoreactive analogue of brain fodrin that is associated with the cell cortex of Dictyostelium amoebae. Cell. Motil. Cytoskel. 11, 303-1 7 13 Wessel, G.M. and Chen, S.W. (1993). Transient. localized accumulation of alpha-spectrin during sea urchin morphogenesis. Dev Blol. 155. 161-71 14 Maude, D., Guillett, G., Rogers, P. and Charest, P. (1991). Identification of a 220 kDa membrane-associated plant cell protein immunologically related to human p-spectrin. FEBS Lett. 294,77-80 15 Sahr, K.E. eta/. (1990). The complete cDNA and polypeptide sequences of human erythroid alpha-spectrin. J. Biol Chem. 265, 4434-43. 16 Moon, R.T. and McMahon, A.P. (1990). Generation of diversity in nonerythroid spectrins. Multiple polypeptides are predicted by sequence analysis of cDNAs encompassing the coding region of human nonerythroid alpha-spectrin. J. B id Chem. 265,4427-33. 17 Wasenius, V.M. eta/. (1989). Primary structure of the brain alpha-spectrin [published erratum appears in J. CellBiol (1989) Mar; 108(3), following 11751. J Cell Biol. 108,79-93. 18 Winkelmann, J.C. eta/. (1990). Full-length sequence of the cDNA for human erythroid beta-spectrin. J. Bid. Chem. 265, 11827-32 19 Hu, R.J., Watanabe, M. and Bennett, V. (1992). Characterization of human brain cDNA encoding the general isoform of beta-spectrin. J. Biol. Chem 267. 18715-22 20 Bloom, M.L., Birkenmeier, C.S. and Barker, J.E. (1 993). Complete nucleotide sequence of the rnurine erythroid beta-spectrin cDNA and tissue-specific expression in normal and jaundiced mice. Blood82.2906-14. 21 Ma, Y. etal. (1993). The complete amino acid sequence for brain beta spectrin (beta fodrin): relationship to globin sequences [published errata appear in Brain Res. Mol. BrainRes. (1993) Oct; 20(1-2),179 and (1994) Jan: 21(1-2).181]. Brain Res. Mol. Brain Res. 18, 87-99 22 Hartwig, J.H. (1994). Actin binding proteins 1: Spectrin superfamily. Protein Profile 1 , 71 1-762. 23 Coleman, T.R., Fishkind, D.J., Mooseker, M.S. and Morrow, J.S. (1989). Functional diversity among spectrin isoforms. Cell. Mofil Cyfoskel. 12.225-47. 24 Castresana, J. and Saraste, M. (1 995). Does Vav bind to F-actin through a CH domain? FEBSLeft. 374, 149-151 25 Djinovic, K., Banuelos, S. and Saraste, M. (1997). Crystal structure of a calponin homology domain. Nature Structural Biology3, 175-1 79. 26 Musacchio, A,, Gibson, T., Lehto, V.-P. and Saraste, M. (1992). SH3-an abundant protein domain in search of a function. FEBS Left 307,55-61. 27 Trave, G., Pastore, A,, Hyvonen, M. and Saraste, M. (1995). The C-terminal domain of alpha-spectrin is structurally related to calmodulin. Eur. J. Biochem 227, 35-42. 28Gibson,T., Hyvonen, M., Musacchio, A., Saraste, M. and Birney, E. (1994). PH domain: the first anniversary. Trends Biochem. Sc;. 19. 349-353. 29 Manno, S., Takakuwa, Y., Nagao, K. and Mohandas, N. (1 995). Modulation of erythrocyte membrane mechanical function by b-spectrin phosphorylation and dephosphorylation. J. Biol. Chern. 270,5659-5665. 30 Hyvonen, M. etal. (1995). Structure of the binding site for inositol phosphates in a PH domain. EM60 J 14, 4676-4685. 31 Leto, T.L., Pleasic, S., Forget, B.G., Benz, E.J. and Marchesi, V.T. (1989). characterization of the calmodulin-binding site of nonerythroid alpha-spectrin. Recombinant protein and model peptide studies J Biol. Chem. 264, 5826-30. 32 Dubreuil, R.R., Brandin, E., Reisberg, J.H., Goldstein, L.S. and Branton, D. (1991). Structure, calmodulin-binding. and calcium-binding properties of recombinant alpha spectrin polypeptides J. Biol. Chem. 266, 7189-93. 33 Speicher, D.W. and Marchesi, V.T. (1984). Erythrocyte spectrin is comprised of many homologous triple helical segments. Nature 31 1 , 177-80. 34 Rost, B. and Sander, C. (1993). Prediction of protein secondary structure at better than 70% accuracy. J. Mol Bid. 232, 584-599. 35 Yan, Y. eta/. (1993). Crystal structure of the repetitive segments of spectrin Science 262, 2027-30. 36 Parry, D.A., Dixon, T.W. and Cohen, C. (1992). Analysis of the three-alpha- helix motif in the spectrin superfamily of proteins Biophys. J 61, 858-67. 37 Lupas, A., van Dyke, M. and Stock, J. (1991). Predicting coiled coils from protein sequences. Science 252, 1 162-1 164. 38 Winograd, E., Hume, D. and Branton, D. (1991) Phasing the conformational unit of spectrin. Proc. Natl Acad. Sci. USA 88. 10788-91.

39 MacDonald, R.I., Musacchio, A., Holmgren, R.A. and Saraste, M. (1994). Invariant tryptophan at a shielded site promotes folding of the conformational unit of spectrin. Roc. NatlAcad. Sci. USA91, 1299.303 40 Lusitani, D.M. eta/. (1 994). The first human alpha-spectrin structural domain begins with serine. J. Biol.Chem. 269,25955-8. 41 Tse, W.T. eta/. (1 990). Point mutation in the beta-spectrin gene associated with alpha 1174 hereditary elliptocytosis. Implications for the mechanism of spectrin dimer self-association. J, Clin. lnvest. 86, 909-1 6. 42 Speicher, D.W. et a/. (1993). Location of the human red cell spectrin tetramer binding site and detection of a related 'closed' hairpin loop dimer using proteolytic footprinting. J. Biol. Chem. 268, 4227-35. 43 Kennedy, S., Weed, S., Forget, B. and Morrow, J. (1994). A partial structural repeat forms the heterodimer self-association site of all b-spectrins. J. Biol. Chem. 269, 11400-1 1408. 44 Sahr, K.E. eta/. (1993). Spectrin cagliari. an Ala+Gly substitution in helix 1 of beta spectrin repeat 17 that severely disrupts the structure and self-association of the erythrocyte spectrin heterodimer. J. Bioi Chem. 268. 22656-62. 45 Parquet, N. et a/. (1994). Identification of three novel spectrin alpha 1174 mutations in hereditary elliptocytosis: further support for a triple-stranded folding unit model of the spectrin heterodirner contact site. 6iood84.303-8. 46 Kotula, L. eta/. (1991). The exon-intron organization of the human erythrocyte a-spectrin gene. Genomics 9, 131 -40. 47 Amin, K., Scarpa, A,, Winkelmann, J., Curtis, P. and Forget, B. (1993). The exon-intron organization of the human erythroid b-spectrin gene. Genomics 18, 1 18-1 25. 48 Sheterline, P., Clayton, J. and Sparrow, J. (1995). Actin Protein Profile2, 1- 68. 49 Gilmore, A.P., Parr, T., Patel, B., Gratzer, W.B. and Critchley, D.R. (1994). Analysis of the phasing of four spectrin-like repeats in alpha-actinin. Eur J Biochem. 225.235-42

50 Winder, S., Gibson, T. and Kendrick-Jones, J. (1995). Dystrophin and utrophin the missing links! FEBSLett. 369. 27-33. 51 Li, X. and Bennett, V. (1 996). Identification of the spectrin subunit and domains required for formation of spectrinladducinlactin complexes. J. Biol. Chem 271, 15695-1 5702. 52 Rybakova, I., Amann, K. and Ervasti, J. (1996). A new model for the interaction of dystrophin with F-actin. J. CeliBiol. 135. 661-672. 53 Jarrett, H. and Foster, J. (1995). Alternate binding of actin and calmodulin to multiple sites on dystrophin. J. Biol. Chem. 270, 5578-5586 54 Corrado, K., Mills, P.L. and Chamberlain, J.S. (1994). Deletion analysis of the dystrophtn-actin binding domain. FEBS Lett. 344,255-260. 55 Kennedy, S.P., Warren, S.L., Forget, B.G. and Morrow, J.S. (1991) Ankyrin binds to the 15th repetitive unit of erythroid and nonerythroid beta-spectrin J. Cell Biol. 115, 267-77. 56 Dubreuil, R. (1991). Structure and evolution of the actin crosslinking proteins BioEssays 13,219-228. 57 Thompson, J.D., Higgins, D.G. and Gibson, T.J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucieic Acids Res. 22.4673-4680.

Jaime Pascual and Matti Saraste' are at the E u r o p q Molecular Biology Laboratory, PO Box 102209, D-69012 Heidelberg, Germany Jose Castresana IS at the Institute of 1 Zoology, University of Munich, PO Box 202136, D-80021 Munich, Germany *Corresponding author (E-mail saraste@embl-Heidelberg de)

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evolution of the spectrin repeat

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